Chapter 13 cont

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Chapter 13 (cont)

• Applications or Recombinant DNA Technology

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Integration What happens in yeast systems with
Yip?

• Two ways that a Yip plasmid might integrate and
  convert a mutant X to WT allele
• NOTE Homologous Recombination (integration).

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Fig 13-13 Promoter bashing in yeast (and other
animals) YIp
Uses a reporter gene Beta galactosidase or
luciferase or any other genes that encode enzymes
that can easily be measured.
Example of promoter bashing experiment 1st Cut
(RE) DNA to be deleted 2nd Exodigest (with time
or enzyme dilutions) 3rd isolate back mutants,
restriction map or sequence various
deletions. 4th test for gene activity.
You would use a non-integrating plasmid for these
experiments!
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Genetic Engineering in Plants

• Commercially very valuable and recomb. DNA adds
  great potential
• Ti Plasmid is primary vector.
• Bacterial host for Ti A. tumefaciens (causes
  crown gall disease (tumors at base of plant)
• Tumors due to plasmid (T-DNA region) which
  integrates randomly into plant genome
• T-DNA encodes opines (gene products) which the
  host bacterium uses.

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Plants as hosts for overexpression-can make ptns
from foreign genes-can make transgenic plants
Ti Plasmid (Fig. 13-15)

• T DNA genes that promote uncontrolled growth in
  tumor
• Large DNA of 200 kb thus not ideal vectors
• To use need a 2nd intermediate vector
• With Ti and 2nd vector, make co-integrate which
  then goes into plants
• Review Fig. 13-16
• Standard methods of using transformation and
  antibiotic selection method

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Transgenic plantsGM foods are a reality

• Putting genes in plants to promote yields, reduce
  insect devastation
• Bacillus thuringensis toxin (Bt toxin) genes
  introduce to plants (damage insect larvae)
  thereby resistant plants made.
• Bt approach very specific (not pesticide based)
  non- health hazard to man
• Ti Plasmid based system used to introduce Bt
  genes

• Molecular Farming Plants used as factories to
  make
• Vaccines in 3rd world..great way to introduce a
  vaccine!
• Pharmaceuticals

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GM foods issues

• Allergic reactions to gene products
• GM plants made to be resistant to
  herbicidesleads to elevated use and exposure to
  toxins in man
• GM plants escape into ecosystem and eat the world

I hate it when that happens
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Genetic Engineering in Animals
Tissue specific promoter
TPA clot buster

• Model systems C. elegans, D. melanogaster, mice
  (read over Drosophila will discuss more later)
• Production of pharmaceuticals in animals
  example of transgenic sheep
• Could be used to make a product (tissue
  plasminogen activator, to dissolve clots) or
  could be a vaccine!

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Gene disruptions in Mice

• The knockout mouse

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Kill TK cells
Rare!
Pretty common
Very common
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If the ES cells take then offspring will be
chimeric and have both black and brown coat color
A agouti, a black, M wt allele, m mutated
(ko) allele
Indicator Coat color. A (agouti or brown) is
dominant. ES (embryonic stem cells) are from an
A mouse.
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Chimera males mated back to non-agouti
females.Southerns or PCR to ID heterozygotes
These are crossed (mated) to produce
homozygotes. The phenotype can be observed.
PhenotypeCurly tail
Problems Embryonic lethals, essential genes for
development, etc.
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GENE THERAPY
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How it works

• Gene replacement reverse of knockout
• Similar to complementation in Benzers T4
• Host functions are supplied by gene delivery
  methods
• Host is genetically cured
• Works in many animals
• Success limited in man

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RGH Correction of Dwarfism in mice Fig 13-23
Regulated promoter
Correcting allele
Only 1 are TG mice
Mendelian dom. pattern
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Types of gene therapy in mammals
Cannot correct defects in man with this approach
(too early)
Germline therapy not likely any time soon in man
Gives permanent change to human gene pool. All
gametes would carry the corrected or modified
gene.
Somatic Changes in the bodies cells that are
not transmitted to offspring (vertical
transmission). Partial liver resection/gene
therapy/replacement with LDLR-/LDLR- patients.
Also Very powerful, HACs Centromere (alpha
satellite seq.), telomeres, rep. Origins. Can
make VERY large artificial chromosomes.
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Detecting mutations how to find problems in
utero, before they surface?
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Chapter 14 Genomics
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• Genomics Study of the molecular organization and
  information content of a genome and its products.
  There are two basic areas
• Structural genomics the physical nature of whole
  genomes
• Functional genomics Characterization of the
  following
• a. The transcriptome the entire range of
  transcripts or primary gene RNA gene products
• b. The proteome the entire array of encoded
  protein gene products.
• GENOMICS became a real field with the complete
  sequencing of genomes (from classical genetic
  maps to sequencing data) and relies heavily on
  computational analyses of DNA, RNA and PTN.
• WHAT YOU CAN DO WITH GENOMICS
• Make predictions about ORF disposition, frequency
  and develop conception of the transcriptome and
  proteome
• Learn more about species relationships and
  evolution (comparative genomics)
• Existence of synteny (conservation of gene
  location with large sequence blocks).
• Learn more about functionally important DNA and
  PTN sequence motifs (the good ones (ie. those
  that work best!) are generally well conserved
• Knowledge of repeat themes in motifs helps
  scientists make accurate predictions regarding
  ORFs, regulatory gene regions (TATA elements) and
  so forth.

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A RIDGE region of increased gene expression
(regions of very high gene density as well)
Regional expression profiles for 23 human
chromosomes show a clustering of highly expressed
genes in RIDGEs. Expression levels are shown as a
moving median with a window size of 39 genes.
There are 74 regions with one or more consecutive
moving medians that have a lower limit of four
times the genomic median 27 of them have a
length of at least 10 consecutive moving medians
(indicated by green bars). From Science 2911289
(2001)